There may be as many as 2800 Zn-Proteins in the proteome, more than 10 times the number of non-heme iron proteins or copper proteins. Besides its key functions in enzyme catalysis and protein folding and stability, Zn2+ plays major roles in normal development and growth, in cancer, in immune response, in neuro-synaptic function etc. In many of these activities, Zn2+ trafficking seems to be involved. In order to observe the cellular distribution of "free" or "accessible" Zn2+ and its perturbation by physiological and pathological stimuli, increasing attention has been given to the use of Sensors that undergo changes in their fluorescent properties in the presence of Zn2+. The most commonly used Sensors, TSQ (N-(6-methoxy-8-quinolyl)-p-toluensulfonamide) and its close relative, Zinquin, reveal a highly asymmetric distribution of intracellular "chelatable" Zn2+ and fluorescence enhancement in response to agents such as nitric oxide donors. It is generally thought that TSQ and Zinquin bind Zn2+ from pools of "free" or modestly bound metal ion to form fluorescent Zn(TSQ)2 or Zn(Zinquin)2. To the contrary, preliminary results are fully consistent with the hypothesis that these Sensors become fluorescent by forming Sensor-Zn-Protein ternary complexes. In addition, model studies with TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine), a cell permeant Zn2+ chelator used to quench Zn-Sensor fluorescence, and nitric oxide, an agent that increases intracellular Zinquin fluorescence, suggest that some of their effects may involve Sensor-Zn-Protein adduct chemistry as well. These findings raise the question with respect to TSQ, Zinquin, and other Zn2+ Sensors, "What is being imaged?" The overall objective of the proposal is to address this question with complementary in vivo and in vitro methods that are both needed to resolve this question. The specific aims are: 1. To establish a set of basic properties that characterizes intracellular imaging with TSQ and Zinquin. 2. To isolate and identify individual proteins to which TSQ is bound, putatively, TSQ-Zn-Protein adducts. 3. To define the cellular and molecular characteristics of the reaction of TPEN with TSQ and Zinquin-treated cells. 4. To investigate the generality of the findings of Specific Aims 1 and 2 in other cell types and conditions. 5. To conduct model studies with TSQ, Zinquin, and other Sensors and a selection of Zn-Proteins. 6. To test the hypothesis that Sensor-Zn-Protein adducts play a significant role in cellular Zn2+ imaging by other Sensors. The major new tool that will be employed in this study is laser ablation-inductively coupled plasma mass spectrometry. It provides the opportunity to locate Zn-Proteins separated within a proteomic background by native polyacrylamide gel electrophoresis. Together with sensitive analysis of Sensor fluorescence and protein location, Sensor-Zn-Proteins can be located and subjected to mass spectral analysis for identification. Variants of this methodology will be used to begin to answer the question raised by Zn2+ Sensor based microscopy: "What is being imaged?" PUBLIC HEALTH RELEVANCE: Zinc is an essential nutrient that plays key roles in normal fetal development, growth, immune central nervous system function, and cancer cell proliferation, among others. Zinc fluorescent Sensors are increasingly used as microscopic probes to study how zinc participates in these processes. Because relatively little is known about how such Sensors image intracellular zinc or what they image, the objective of this proposal is to understand the chemistry underlying microscopic fluorescent imaging by commonly used zinc Sensors.